EP1910831B1 - Verfahren und system zur konzentrationsbestimmung eines analyt-enzym-komplexes oder analyt-enzym-konjugats, insbesondere zur elektrochemischen detektion des analyten - Google Patents

Verfahren und system zur konzentrationsbestimmung eines analyt-enzym-komplexes oder analyt-enzym-konjugats, insbesondere zur elektrochemischen detektion des analyten Download PDF

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EP1910831B1
EP1910831B1 EP06778077.5A EP06778077A EP1910831B1 EP 1910831 B1 EP1910831 B1 EP 1910831B1 EP 06778077 A EP06778077 A EP 06778077A EP 1910831 B1 EP1910831 B1 EP 1910831B1
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analyte
enzyme
electrode
concentration
phase
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French (fr)
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EP1910831A2 (de
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Heike Barlag
Konrad Mund
Walter Gumbrecht
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Siemens AG
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Siemens AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/817—Enzyme or microbe electrode

Definitions

  • the invention relates to a method and a system for determining the concentration of an analyte-enzyme complex or analyte-enzyme conjugate, in particular for the electrochemical detection of the analyte.
  • a substrate is converted via the enzyme by hydrolytic cleavage to a reporter molecule whose concentration or concentration change in the static electrolyte is determined by an amperometric measurement method based on the oxidation and reduction of the reporter molecule on the same noble metal electrode.
  • Biosensors are used to determine antibodies, antigens or DNA in blood, water, air or food. They are based on the specific binding of the analyte with a capture molecule, e.g. an antibody with an antigen or a DNA sequence with the complementary DNA capture sequence. The detection of this binding event is often done by optical methods. In this case, fluorescent dyes, e.g. inserted into the analyte during a DNA analysis by the PCR and later read out at the positions with different capture sequences.
  • fluorescent dyes e.g. inserted into the analyte during a DNA analysis by the PCR and later read out at the positions with different capture sequences.
  • optical systems are expensive and expensive to handle. The required equipment is sensitive and not suitable for field tests.
  • Biosensors Here, the detection is carried out by the implementation of an electrochemical substance. Widespread electrochemical detection is e.g. at the glucose sensors. The glucose is oxidized by a redox enzyme, the glucose oxidase, and at the same time the oxygen present is reduced. The resulting hydrogen peroxide is then electrochemically oxidized again and so amperometrically determines the glucose concentration. In further developments of these glucose sensors, mediators replace the oxygen. In the oxidation of glucose by the redox enzyme, the mediator, e.g. 1,1-dimethylferrocene, reduced.
  • the mediator e.g. 1,1-dimethylferrocene
  • the electrochemical oxidation of the enzymatically reduced mediator for amperometric determination of the glucose concentration can take place here at significantly lower potentials, making the measurement more accurate and less prone to failure.
  • numerous amperometric pulse methods have been developed that did not determine the total amount of redox mediator, but only the previously enzymatically reduced portion.
  • the absolute amount of a redox-active substance must be measured as sensitively as possible.
  • the actual marker for the presence of the analyte at a sensor position is the enzyme itself.
  • a biotin marker is added to the analyte in the PCR.
  • On a biosensor different DNA capture sequences are bound to the different sensor positions.
  • the analyte only hybridizes to the appropriate sequences and the unbound analyte is washed away.
  • a streptavidin enzyme eg alkaline phosphatase, is now bound to the biotin marker molecule. If enzyme substrate, for example p-aminophenyl phosphate, is added, p-aminophenol is liberated only at the sensor positions to which the analyte has bound due to the hydrolytic cleavage of the phosphate.
  • p-aminophenyl phosphate as a substrate for the alkaline phosphatase is described in CLIN. CHEM. 36/11, p.1941-1944 (1990 ), where a bead-based immunoassay is described. Whether analyte has bound to the bead-immobilized antibodies is also indicated here by alkaline phosphatase.
  • the beads are incubated with p-aminophenyl phosphate and then the supernatant solution analyzed for p-aminophenyl phosphate in a flow injection analysis system. This solution flows through an electrochemical sensor whose working electrode is constant at approx. +0.1 V vs. approx. Ag / AgCl reference electrode is polarized.
  • the measurement in the flowing electrolyte or sample volume has the advantage that no appreciable depletion of the p-aminophenol takes place in front of the electrode.
  • the river is constantly replenished.
  • Such a system is not microsystem capable.
  • the electrochemical sensor can read only one sample at a time and the required volumes are large.
  • electrochemical detection must be performed in the quiescent electrolyte so that the sensors only detect the signal from the scavengers immobilized directly on it.
  • the presence of the enzyme is indicated by an increase in the p-aminophenol concentration.
  • Simple electrochemical sensors, where the working electrode is at a constant potential, are not suitable for this purpose. Due to the ongoing implementation of the enzyme product this is consumed. The increase of the concentration by the enzyme activity is thus superimposed by a decrease of the concentration by the measurement itself.
  • each sensor consists of two interdigital electrodes.
  • one of the electrodes becomes positive and the others negatively polarized.
  • the p-aminophenol is oxidized at the first electrode and thus consumed. If it can now diffuse to the second electrode, it is again reduced there and is available again for the measurement at the first electrode.
  • the system should not be convective, ie neither electrode nor solution should be stirred or moved and the measurement frequency ⁇ 1 Hz.
  • an associated measuring device is to be created.
  • the invention it is proposed to pulse the potential of the working electrode, which has already been proposed in another context.
  • measurement phases and relaxation phases are now alternately formed in the present invention, wherein the measurement phase pulse lengths are chosen so that towards the end of the pulse, the capacitive current is small compared to the Faraday current, and wherein the relaxation phase pulse lengths are chosen in that towards the end of the pulse the concentration gradient is relaxed, so that at the beginning of the following measuring phase the concentration change of the redox-active enzyme product - caused by the consumption of the enzyme product by the measurement itself - is largely reversed.
  • the current measured at the end of the measuring phase constitutes a significant measuring signal, which is not to be expected 'a priori'.
  • the rapid measurement of the concentration and in particular the change in concentration of an enzyme product in molecular biological detection systems by means of the electrochemical redox reaction of the enzymatically formed redox-active substance in the anchored on a chip belt in caverns working electrodes by a cyclic pulse loading, which measured after the transhipment of the double layer Current forms the measuring signal.
  • the invention is based on the finding that, with a suitable measuring method for determining the concentration of a redox-active substance, it is advantageously possible to measure electrodes with diameters in the order of magnitude of a few 100 ⁇ m up to 1 cm. Especially complex Interdigital electrodes are no longer necessary.
  • low-cost transducer arrays can also be used, as described in detail, for example, in the non-prepublished German patent application AZ. 10 2004 004 654.9-52 the applicant are described.
  • the measuring structure is simplified compared to the Redoxcycling in that no Bipotentiostat is needed.
  • a simple potentiostat in combination with a pulse generator is sufficient.
  • the potential of the working electrode is pulsed.
  • a diffusion layer forms, the thickness of which reaches a maximum value at the end of the measuring period, which depends on the length of the measuring phase.
  • the diffusion layer is broken down over time and then degraded again. In both cases, this sets a diffusion layer thickness limited at least in its maximum value. If a reduction is to be observed, the reduction potential must be set during the measurement phase and the corresponding oxidation potential during the relaxation phase.
  • FIG. 5 are the associated signal curves for the basis of the FIGS. 1 to 4 defined FRA method shown. Subsequently, based on the FIGS. 6 to 12 Reflections on the accuracy of the new measurement method is reproduced and is finally in FIG. 13 reproduced a concrete measuring device.
  • an exemplary transducer array whose structure in the Figures 14 and 15 shown. Another transducer array is shown in FIG. 16.
  • FIG. 17 finally gives the procedure in the application of the arrangement as a DNA sensor again.
  • Reference number 1 denotes a support for at least one measuring electrode 2, which is assigned a current path 3 for an electron flow, wherein an analyte 4 is present.
  • an enzyme 5 whose concentration is a measure of the concentration of the analyte 4 by being directly or indirectly bound to the analyte 4 or displaced by the analyte 4.
  • FIGS. 3 and 4 For binding to the carrier 1 is in FIGS. 3 and 4 a catcher 6 indicated.
  • Reference numeral 7 denotes a substrate which is reacted by the enzyme 5 by means of hydrolytic cleavage, resulting in a reporter molecule 8 which can be reversibly electrochemically reacted under conditions in which the substrate 7 is stable.
  • the arrangement of carrier 1 and electrode 2 represents a sensor.
  • the electrode 2 is polarized for the duration of a measuring phase A in such a way that the reporter molecule 8 is oxidized or reduced, and polarized for the duration of a relaxation phase B in such a way that the oxidized resp , reduced form of the reporter molecule 8 at the same electrode 2 is again reduced or oxidized. That is through the FIGS. 3 and 4 clarified.
  • the current flowing during the measurement phase A is a measure of the concentration of the substrate 7 reacted by the enzyme 5.
  • the analyte contains a labeling element and if the enzyme contains a coupling element which specifically binds to the labeling element of the analyte.
  • the enzyme can also be bound directly to the analyte.
  • a capture molecule is bound in known manner on or in the vicinity of an electrode, which binds specifically to the analyte.
  • the different phases are marked during the measurement. Which the measuring and which the Relaxation phase depends on whether an oxidation or reduction current is to be measured.
  • the pulse lengths for oxidation and reduction need not be the same, ie the times t Ox and t Red may differ. Also, the potentials ⁇ Ox and ⁇ Red need not be symmetrical to the redox potential ⁇ 0 of the species.
  • FIG. 5 shows the potential curve using rectangular pulses.
  • the potentials ⁇ Ox or ⁇ RED and the times ⁇ t ox or ⁇ t RED with the pulse shape 11 are plotted in arbitrary units.
  • the pulse shape 11 with the pulse lengths for oxidation and reduction need not be symmetrical. It is also possible to realize forced ralaxation amperometry with triangular voltage curves or sinusoids.
  • the measuring phase is denoted by A and the relaxation phase by B.
  • the potential curve 11 are still a curve 12 for the concentration of
  • Enzyme product in front of the electrode and additionally the electric current 13 shown as a measurement signal is therefore always at the end of the relaxation interval and is a measure of the relaxed change in the change in the crown concentration.
  • the concentration of a molecule present in its reduced form should be measured.
  • the potential is positive relative to the redox potential of the species.
  • the highest measuring currents are achieved when the potential is so far in the positive that sets a diffusion limit current.
  • the current is then not limited by the kinetics of the redox reaction, but only by the diffusion.
  • a further increase in the measurement signal can be achieved by limiting the electrolyte space in front of the electrode.
  • the reporter molecule formed by the enzyme diffuses only in part to the electrode. The other part diffuses into the electrolyte space facing away from the electrode.
  • saturation of the electrolyte space with reporter molecule takes place during the measurement phase, and each further formed reporter molecule increases the concentration in front of the electrode. This applies to the total concentration of reduced and oxidized reporter molecule.
  • it is difficult to carry out the biochemical assay with a very small electrolyte space this results in the requirement for an electrolyte chamber with a variable height. Only at the beginning of the measurement of the electrolyte space is reduced and thus increases the sensitivity of the sensor.
  • the diffusion layer After 0.1 s, the diffusion layer has a thickness of about 25 ⁇ m. After 0.25 s, the extent of the pAP-depleted layer is already 40 ⁇ m. The thicker this layer is, the longer the relaxation will take by diffusion.
  • the FIG. 7 shows the current density as a function of the pAP concentration in the solution for different pulse sequences, which are characterized by curves 31 to 34.
  • the measurement phase is always 0.25 s, the relaxation phase 0.75 s.
  • the current measurement takes place 0.24 s after the beginning of the measuring phase.
  • the oxidation potential during the measurement phase is +200 mV related to the redox potential.
  • the potential was varied during the relaxation phase. It takes values between -300 mV and 0 mV based on the redox potential.
  • ⁇ red / ⁇ ox at characteristic 31-300 mV / 200 mV, at characteristic 32 -200 mV / 200 mV, at characteristic 33 -100 mV / 200 mV and at characteristic 34 0 mV / 200 mV.
  • the current density set after a longer measuring time was used.
  • the measurement is performed but so that the solution is first stirred or pumped.
  • the pAP formed by the enzyme is thereby washed away and a constant background current sets in.
  • the pump is stopped and the increasing concentration measured during the first few seconds.
  • Typical slopes are on the order of 2 ⁇ A / cm 2 s. If the measurement itself leads to a decrease in the signal, then both effects are superimposed and an excessively low slope of the current and thus enzyme activity are measured. Since this decrease in the current through the consumption of the substance also depends on its concentration, this effect can not be eliminated by normalizations.
  • the concentration was 50 ⁇ M pAP, the potential during the measurement phase +200 mV.
  • the duration of the measurement phase was 250 ms, with the current measurement taking place after 240 ms.
  • the potential during the relaxation phase was 0V in a first experiment. ⁇ 0 , in a second experiment-300 mV vs. ⁇ 0 .
  • the duration of the relaxation phase was varied between 250 ms and 4.75 s.
  • FIG. 9 is a graph showing the dependence of the current density j of the time: there are curves 51 to 54 for different relaxation phase durations .DELTA.t Red , specifically between 0.255 and 4.755.
  • the currents drop sharply within the first 10 seconds of the measurement.
  • the decrease is 14 ⁇ A / cm 2 in 10 s.
  • the duration of the relaxation phase is increased to 4.75 s, the decrease of the signal to 9 ⁇ A / cm 2 in 10 s decreases.
  • the shorter the duration of the relaxation phase the stronger this decrease in the signal over time. But even with long relaxation times, the decrease of 0.9 ⁇ A / cm 2 s is still significant compared to the slopes measured in the order of 2 ⁇ A / cm 2 s in use should.
  • the signal drop With a relaxation time of 0.25 s, the signal drop is still 8 ⁇ A / cm 2 in 10 s. With a relaxation time of 0.75 s, this value is still 2 ⁇ A / cm 2 in 10 s, at 1.75 still 1 ⁇ A / cm 2 in 10 s and for 4.75 s only 0.5 ⁇ A / cm 2 in 10 s. Even with a relaxation time of 0.75 s, ie a measurement frequency of 1 Hz, and a relaxation potential of-300 mV, the signal drop and thus also the error is only about 1% of the expected measured value.
  • the experiments show the influence of duration and potential of the relaxation phase on the measurement signal. Further information about the effect of forced relaxation amperometry can be obtained from simulation calculations. On the one hand, the current density during redox cycling is calculated, on the other hand the current density without redox cycling is determined for comparison.
  • the electrolyte space has a thickness of 100 ⁇ m. Both the oxidation and the reduction potential are chosen such that the reaction takes place in the diffusion limit current range, ie the currents are maximum.
  • the pulse lengths are 250 ms for the oxidation potential and 750 ms for the reduction potential.
  • the parameters were the same except for the fact that no potential is given during the relaxation phase and no current can flow through the potentiostat. During this time, the system is therefore decoupled electrochemically.
  • the y-axis sections of the simulation data were adjusted to the experimental results.
  • FIG. 11 shows a good agreement of experiment and simulation for the forced relaxation amperometry, where 71 represent the measured values and 72 the calculated characteristics.
  • the decrease in the current density is low under these conditions with 2 ⁇ A / cm 2 in 4 s.
  • the current density drop during the first 4 s is already 12 ⁇ A / cm 2 , which is illustrated by the characteristic curve 73.
  • the results for a measuring method with potentiostation during the relaxation phase are at the redox potential, which is represented by characteristic curve 74 ,
  • the improvement of the signal constancy by a factor of 6 can be directly attributed to the concentration profiles.
  • the following figure shows the calculated concentration profiles of pAP as a function of the distance from the electrode as they appear at the end of the 5th relaxation phase.
  • Characteristic curve 82 for a measurement without redox cycling in the practice of measuring the concentration of a substance that can be oxidized but whose oxidation products are not reducible again. The same would be the case for a substance that reduces, but whose reduction products can not be oxidized again. In the case of a biochemical sensor, this could be, for example, naphthol, which, like the pAP, can be released in an enzymatic reaction.
  • the measuring device is shown in detail: Except by a transducer array 100, which is based on the Figures 14 and 15 will be described in detail, the measuring device is essentially realized by a suitable potentiostat 105 in combination with a pulse generator 106 which optionally provides square wave, triangular or sinusoidal pulses.
  • a pulse generator 106 which optionally provides square wave, triangular or sinusoidal pulses.
  • the potentiostat 105 is designed such that suitable potentials are provided.
  • the pulse length, the repetition rate and the level of the potential can be specified here.
  • the pulse lengths of the measurement phases and the relaxation phases can be set separately and be of different lengths.
  • the potentials can also vary.
  • the signal of the potentiostat 105 is applied to an in FIG. 9 not shown in detail signal processing unit connected, with which an evaluation taking into account the above comments on the measurement methodology and accuracy. In general, the results in FIG. 13 shown as I out Signal curve for evaluation.
  • the transducer array 100 is reproduced as part of the measuring device, which is planar and flexible and in particular is inexpensive to produce. It is essential that now with the forced relaxation amperometry with a simplified transducer array 100 can be measured.
  • the Figures 14 and 15 show the top and bottom of the transducer array 100 - consisting of a metal substrate 101 and an insulator layer 102. On the top, for example, circular recesses 103 i , which are referred to as cavities, shown.
  • the cavities 103 i are formed by the structuring of the insulator 102.
  • the upper side of the metal substrate is exposed and forms a measuring point if an analyte is applied.
  • the representation of the back shows by structuring Structuring and thus a separation of the metal substrate 101 into mutually insulated parts.
  • Each metal island corresponds to a depression 103 i on the front side. By points, the possible contact points for a so-called. Needle card for simplified electrical contacting of the metal surfaces are indicated. It is essential that several metal islands, preferably three, define a sensor with an analyte and are suitable for carrying out electrochemical measurements with the associated electrodes, which form a measuring electrode WE, a counter electrode GE and a reference electrode RE.
  • FIG. 16 are mounted on the support 1 with sensitive surface or the individual sensitive elements discrete electrical contacts.
  • the contacts form inputs for the electrical measuring circuit.
  • two supply voltage inputs V dd , V ss , a ground potential input GND, an input for a clock signal, an input V in for a control voltage, and an input for a reset signal are present.
  • a multiplexer 210, a "gray counter &decoder" 215 and an amplifier 220 are integrated on the chip 1 by means of standard silicon technology.
  • the measurement signal is detected at the output 'out', wherein in an array arrangement with the plurality of cavities as mxn individual sensors, a multiplex signal is obtained, which is read, for example, with a frequency of 10 kHz.
  • the multiplex signal output on a single line 'out' consists of a pattern of discrete voltage values, from which the individual sensor signals are obtained by means of a de-multiplexer in an evaluation unit.
  • FIG. 16 In an alternative measuring arrangement instead of the cantilevered and flexible transducer array according to the FIGS. 13 to 15 a transducer array used in thin-film technology on a rigid substrate.
  • flat electrodes are present, which have an extension which is greater than the diffusion length.
  • a typical diffusion length is 25 ⁇ m for the example given above, so that the flat electrodes have an extent ⁇ 30 ⁇ m, preferably ⁇ 50 ⁇ m.
  • the rigid substrate is in particular silicon, which is preferably provided with an insulator layer.
  • FIG. 14 / 15 a biochemical sensor for DNA analysis is realized Figures 14 / 15 described transducer array 100 of a metal layer and an associated insulator layer with cavities 3 i used.
  • the diameter of the cavities 3 i is 0.8 mm, the depth 90 microns and the distance between two adjacent measuring points 1mm.
  • the electrode surfaces are covered with a 2.3 ⁇ m thick gold layer.
  • the height of the electrolyte space above the transducer array may be varied during the assay.
  • the electrolyte space over the transducer array can be limited by a flexible material, wherein the material can be pressed by a force acting from above in the direction of the array.
  • the electrolyte space is reduced to the extent that the transport of transporter molecules between the sensors of the array is suppressed.
  • the sensor arrangement thus consists of at least three, but advantageously also of four electrodes.
  • One of the electrodes is then occupied as reference electrode with a silver / silver chloride (Ag / AgCl) layer, another electrode is used as the counter electrode CE and the two other electrodes serve as measuring electrodes WE.
  • oligonucleotide sequence of length 25 anchored by means of a terminal thiol group on the gold surface as a positive sample.
  • the second measuring electrode remains free as a negative sample.
  • both surfaces were incubated with a solution of 1 mg bovine serum albumin per ml for 15 minutes and then the sensor array was inserted into a 100 ⁇ m deep flow channel.
  • 10 ⁇ l of a 10 pM biotinilated target sequence are pumped over the electrodes in about 5 minutes. Then, after a washing step, a solution of streptavidin labeled alkaline phosphatase is added.
  • the washing is carried out with a buffer solution of 100 mM tris (hydroxymethyl) aminomethane titrated to pH 8 with hydrochloric acid and 130 mM NaCl.
  • a 2 mM solution of the enzyme substrate para-aminophenyl phosphate (pAPP) in the buffer solution is pumped over the sensor array.
  • the enzyme substrate pAPP is converted to para-aminophenol (pAP).
  • reference electrode RE For measurement are reference electrode RE, counter electrode CE and each one of the two measuring electrodes WE are each connected in a three-electrode arrangement to a potentiostat.
  • the measurement is carried out by means of "forced relaxation amperometry".
  • the para-amino phenol formed by the enzyme is oxidized to quinone imine.
  • the oxidation potential ⁇ Ox is +200 mV vs.. ⁇ 0 .
  • the pulse length of the measuring phase is 250 ms, that of the relaxation phase 750 ms.
  • the current measurement takes place 240 ms after the beginning of the measuring phase.
  • the positive sample ie the electrode with the capture sequence is connected.
  • the solution with the enzyme substrate flows - conveyed by a pump - first over the negative sample and then over the Positive sample.
  • pAP formed by the enzyme is washed away by the electrodes, so that when the pump is switched on, the current is constant and low. If the pump is stopped, the pAP concentration increases over time due to the enzyme activity. In the measurement this is shown by a strong increase of the current signal with 20 nA / s. When the pump is switched on again, the signal drops back to the original value. This process can be repeated as often as you like.
  • the FIG. 17 shows the temporal course of the current at pump "on” / "stop” on the described sensor arrangement with positive and negative sample:
  • the characteristic curve 121 shows the course of the pump current.
  • the result for the experiment is a specific course with individual peaks, where parameter is the activation of the pump on the one hand ("stop” / "on") and the switching of the measuring electrodes on the other.
  • the measuring range of interest is underlined with a dashed line.
  • t 400 s was switched to the negative sample.
  • the current first drops when the pump stops, then remains constant for a short time and then rises slowly. This increase is caused by the diffusion of pAP from the positive to the negative sample.
  • pump "on” a peak current is added because the electrolyte first flows from the positive to the negative sample and thus transports an increased pAP concentration to the adjacent electrode. Overall, the discrimination of positive and negative samples is very good.

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EP06778077.5A 2005-08-04 2006-07-31 Verfahren und system zur konzentrationsbestimmung eines analyt-enzym-komplexes oder analyt-enzym-konjugats, insbesondere zur elektrochemischen detektion des analyten Active EP1910831B1 (de)

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DE102005037436A DE102005037436A1 (de) 2005-08-04 2005-08-04 Verfahren und System zur Konzentrationsbestimmung eines Analyt-Enzym-Komplexes oder Analyt-Enzym-Konjugats, insbesondere zur elektrochemischen Detektion des Analyten, und zugehörige Messvorrichtung
PCT/EP2006/064842 WO2007014931A2 (de) 2005-08-04 2006-07-31 Verfahren und system zur konzentrationsbestimmung eines analyt-enzym-komplexes oder analyt-enzym-konjugats, insbesondere zur elektrochemischen detektion des analyten, und zugehörige messvorrichtung

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US20100155263A1 (en) 2010-06-24
CN101283277B (zh) 2012-08-29
ES2446241T3 (es) 2014-03-06
WO2007014931A3 (de) 2007-06-21
DE102005037436A1 (de) 2007-02-15

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